Hepatitis B virus (HBV) is a small DNA virus that causes acute and chronic hepatitis. HBV replicates through an RNA intermediate. Despite an effective vaccine, the 400 million people who are chronically infected with HBV have a 100 fold elevated risk of developing hepatocellular carcinoma. Current treatments are effective in ~50% of cases. The goal of this proposal is to develop novel HBV therapeutics and to test them in cell culture and small animal models of HBV. We hypothesize that we can eliminate HBV in these models by using a combination approach that targets both HBV RNAs and DNAs. We will test several hypotheses regarding inhibition of HBV: 1) Chimeric nucleases composed of a zinc-finger DNA binding domain fused to the catalytic domain of the Fokl endonuclease (ZFPNs) can be designed to specifically bind to HBV genomic DNA and cleave it. Engineered ZFPNs have been used to create double-stranded breaks in the genomic DNA of cultured cells and their binding specificity can be altered by rational design. However, to date, ZFPNs have not been used to cleave episomal viral genomes, nor has their activity been evaluated in mice. We will design ZFPNs that bind and cleave HBV DNAs and evaluate HBV DNA binding and cleavage in vitro as well as DNA cleavage in cultured cells. 2) Thermodynamically optimized HBV RNA interference (RNAi) triggers expressed in the context of an endogenous microRNA will be efficiently processed and incorporated into the silencing machinery, leading to more potent and specific RNAi against HBV. Previously, we used RNA interference (RNAi) to degrade HBV RNAs in mice, with concomitant decreases in viral proteins and replicated DNA genomes. We will utilize recent mechanistic insights to improve incorporation of siRNAs into the silencing machinery. Also, embedding RNAi triggers within a microRNA context will enhance nuclear export and allow regulated and tissue specific polymerase II expression. We will evaluate the potency and specificity of improved RNAi triggers in cultured cells. 3) Adeno-associated virus serotype 8 (AAV8) vectors will deliver HBV ZFPNs and RNAi to the majority of hepatocytes in HBV mouse models, leading to elimination of HBV DNAs and RNAs. AAV8 vectors have been shown to transduce nearly 100% of hepatocytes in mice and are thus an attractive vehicle for delivering HBV therapeutics. We will express HBV RNAi and ZFPNs using AAV8 vectors and test them in HBV mouse models. HBV is the 9th leading cause of death worldwide according to the World Health Organization. New HBV therapeutics could have a major impact on global health. The approaches outlined in this proposal are generally applicable because they act in a sequence-specific manner at the level of DNA and RNA. Thus, they can be adapted to the treatment of diverse infections and diseases.